Energy-dependent proteolytic machines degrade abnormal proteins and many short-lived regulatory proteins. These machines, which fall into three archetypal groups called the 26S proteasome, Clp and Lon, share a common barrel like architecture when viewed in the electron microscope and a common mechanism. In Lon, the ATPase and proteolytic activities are coded within a single amino acid sequence, making substrate recognition and proteolysis a highly coupled process. In Clp the proteolytic core, ClpP, and ATPase component, CIpA, ClpX, or in some bacteria ClpC, are coded for on two separate amino acid sequences and are stable homo-oligomers that can be purified characterized independently. The same is true for HlsUV, which contains a proteolytic component, HslV that is homologous to the n-type subunits of the 26S proteasome, and a Clp-like ATPase, HslU. Thus, CIp and HslUV systems have a major advantage over the Lon as model systems for addressing mechanistic questions, and Dr. Flanagan has exploited this property in his proposal.This proposal is focused on biochemical and x-ray crystallographic studies of ClpX and HslU and their complexes with their respective proteolytic subunits and substrates to examine how these ATPase subunits bind, unfold and translocate substrates into the proteolytic subunits for degradation. These ATPases have a chaperone activity alone that converts to an unfoldase activity when in a complex with their proteolytic component. Thus features of this class of ATPase are likely to have a key role in cellular protein quality control. The principal investigator has additionally proposed experiments to understand how the homo-oligomeric proteolytic subunits can cleave peptide bonds with little apparent sequence specificity; this latter feature is a hallmark of all of the energy-dependent proteases. The experiments proposed herein will allow an overall understanding of the mechanism of energy dependent proteolysis and thus one can expect a deeper understanding of the process of protein quality control which is a essential process in all cells.